The ultimate goal of this research project is to develop a novel injectable, bilayered, biodegradable hydrogel composite for the co-delivery of chondrogenic growth factors and mesenchymal stem cells (MSCs) to influence the degree and quality of cartilage tissue regeneration within osteochondral defects. We hypothesize that controlled dual delivery of transforming growth factor-21 (TGF-21) and insulin-like growth factor-1 (IGF-1) using optimal release kinetics and doses will induce chondrogenic differentiation of progenitor cells within the recipient to influence the regeneration of cartilage tissue in an osteochondral defect. Additionally, we hypothesize that the duration of exposure of MSCs to TGF-21 and osteogenic medium supplements during in vitro expansion will modulate the chondrogenic and osteogenic differentiation stages of the cells, respectively, which will in turn influence the degree and quality of osteochondral tissue regeneration when the cells are encapsulated within and transplanted with a hydrogel construct. Finally, we hypothesize that the co-delivery of growth factor(s) from hydrogel composites, coupled with the transplantation of progenitor cells encapsulated within the hydrogels will act cooperatively to promote regeneration of cartilage tissue in an osteochondral defect, with the initial cell seeding density influencing the degree and quality of the cartilage regeneration. To address these hypotheses, three Specific Aims are proposed. First, TGF-21 and IGF-1 will be loaded into OPF hydrogel constructs at different doses and released with different kinetics to determine the effect of these parameters on tissue regeneration in a rabbit osteochondral defect. Second, MSCs will be exposed to TGF-21 as a chondrogenic culture medium supplement or osteogenic medium supplements for various durations to result in cells of different chondrogenic and osteogenic differentiation stages, respectively, then they will be encapsulated within and transplanted with OPF hydrogel scaffolds (without loaded growth factors) into a rabbit osteochondral defect model to assess the effect of the differentiation stages of the transplanted cells upon osteochondral tissue regeneration. Third, cells of the optimal differentiation stages will be encapsulated for transplantation within OPF scaffolds corresponding to the optimal growth factor delivery formulation and will be implanted into rabbit osteochondral defects to determine the optimal seeding density of the progenitor cells for osteochondral tissue regeneration, which will be assessed post-implantation through histomorphometric analysis and mechanical testing. This novel strategy for the concurrent and spatially defined delivery of chondrogenic growth factors and in vitro expanded autologous progenitor cells to osteochondral defects presents tremendous potential for clinical translation and osteochondral tissue regeneration.